Lytic enzymes such as chitinase and β-1,3-glucanase are known as plant proteins having antifungal or antimicrobial activity against plant pathogenic microorganisms. In vitro experiments have shown that while these types of enzymes will exert their effect if employed alone (Schlumbaum et al. (1986), Nature 324, pp. 365-367; Broglie et al. (1991), Science 254, pp. 1194-1197), if a combination of two or more such enzymes is employed, an enhanced effect can generally be obtained (Mauch et al. (1988), Plant Physiol. 88, pp. 936-942; Sela-Buurlage et al. (1993), Plant Physlol. 101, pp. 857-863). If used to inhibit fungal growth, it is known that these lytic enzymes are required to be used at a concentration of from about several ten to several hundred μg/ml when used alone, or about several μg/ml per enzyme when used in combination. However, so far as the present inventors are aware, none of these lytic enzymes has been demonstrated to exert any antimicrobial effect against Pyricularia oryzae which causes extensive damage to rice crops.
Antifungal peptides (AFP) of low molecular weight exemplified by defensin, also have antimicrobial activity and, among them, it is reported that Ca-AMP1 (Japanese domestic announcement No. 505048/96) and CB-1 (Oita et al. (1996), Biosci. Biotech. Biochem. 60, pp. 481-483) show antimicrobial activity against both Pyricularia oryzae and Rhizoctonia solani. While Rs-AFP1 and Ar-AFP2 (Terras et al. 1992, J. Biol. Chem. 267, pp. 15301-15309), and Ace-AMP1 (Japanese domestic announcement No. 501424/97) show antimicrobial activity against Pyricularia oryzae. These low-molecular weight peptides inhibit 50% of the growth of plant pathogenic microorganisms including the ones mentioned above at a concentration of several μg/ml.
Also, attempts have been made to isolate lytic enzyme genes or low-molecular weight antimicrobial peptide genes and transfer these genes into plants to thereby construct plants tolerant to injury from disease (Broglie et al. (1991), Science 254, pp. 1194-1197; Zhu et al. (1994), Bio/Technology 12, pp. 807-812; Lin et al. (1995), Bio/Technology 13, pp. 686-691; Terras et al. (1995). The Plant Cell 7, pp. 573-588). However hardly any plants to which tolerance at a practically acceptable level is imparted have hitherto been obtained. One reason for this is considered to be that the transferred genes are expressed at a low level only. However, a more fundamental reason is considered to be that antimicrobial proteins per se reported so far have poor antimicrobial activity. Consequently, it has been desired to identify and utilize an antimicrobial protein which is superior in antimicrobial activity to those of the prior art.